1. Field of the Invention
The present invention relates to assays for and treatment of tumors using Bax degradation activity. More specifically, the present invention relates to the using determinations of Bax degredation levels for prognosis and treatment of cancer.
2. Description of Related Art
Apoptosis, a morphologically distinct form of programmed cell death, plays a major role in development, homeostasis, and many diseases including cancer (Song and Steller, 1999). The process of apoptosis can be divided into three fundamental steps: initiation, commitment, and execution (Reed, 1997). The cell death pathway can be initiated by many stimuli and insults, including deprivation of growth factors or treatment with radiation, chemotherapeutic agents or the kinase inhibitor staurosporin (Reed, 1997).
The molecular mechanisms controlling apoptotic commitment are unclear. Results from the most recent experiments have suggested that cellular fate can be determined by Bcl-2 family proteins that are localized in mitochondria (Green and Reed, 1998; Adams and Cory, 1998; Gross et al., 1999). Apoptotic execution is initiated by activation of effector caspase protease (such as caspase-3) (Thornberry and Lazebnik, 1998), which in turn cleaves important cellular proteins, including a poly(ADP-ribose) polymerase (PARP) (Lazebnik et al., 1994), lamin (Lazebnik et al., 1995), DNA-dependent protein kinase (Song et al., 1996) and retinoblastoma protein (RB) (An and Dou, 1996); Tan et al., 1997). The active caspase-3 also cleaves a caspase-activated deoxyribonuclease inhibitor, resulting activation of the deoxyribonuclease that is responsible for the internucleosomal fragmentation of DNA (Enari et al., 1998), a hallmark of apoptotic execution (Thornberry and Lazebnik, 1998).
Several members of the Bcl-2 family (such as Bax, Bid and Bad) promote apoptosis, whereas the other Bcl-2 members (such as Bcl-2 and Bcl-XL) inhibit the cell death process (Green and Reed, 1998; Adams and Cory, 1998; Gross et al., 1999). The Bcl-2 family proteins also can form homodimers or heterodimers. The ratio of pro-apoptotic to antiapoptotic proteins in the Bcl-2 family is involved in determination of cellular fate (Green and Reed, 1998; Adams and Cory, 1998; Gross et al., 1999). In addition to their ratios, the mitochondrial localization of the Bcl-2 family proteins seems essential for their functions. It has been found that the pro-apoptotic Bcl-2 family members promote, while the antiapoptotic members block, the release of cytochrome c from mitochondria to the cytosol (Green and Reed, 1998; Adams and Cory, 1998; Gross et al., 1999). Once in cytosol, the released cytochrome c, together with Apaf-1, binds and activates caspase-9, which in turn cleaves and activates the effector caspase-3 (Li et al., 1997). The three-dimensional structures of Bcl-XL and Bid suggest that these proteins contain domains similar to the pore forming domains of some type of bacterial toxins (Chou et al., 1999). When added to synthetic membranes, Bcl-2, BclXL and Bax were able to form ion channels (Schlesinger et al., 1997; Minn et al., 1997). However, it is unclear whether Bcl-2 family proteins also modulate the pore formation in mitochondria in vivo to mediate cytochrome c release.
In the absence of a death signal, most of the anti- and pro-apoptotic Bcl-2 members are localized in separate subcellular compartments. While pro-apoptotic members mainly remain in cytosol, antiapoptotic members are localized on membranes of mitochondria, endoplasmic reticulum, and nucleus (Gross et al., 1999; Porter, 1999). Following a death signal, the pro-apoptotic members undergo a post-translational modification and/or a conformational change, followed by translocation to membranes of cellular compartments, especially mitochondria (Gross et al., 1999; Porter, 1999). For example, during tumor necrosis factor xcex1- or Fas-induced apoptosis, Bid is first cleaved at its N-terminus by caspase-8, and the carboxy-terminal fragment of Bid is then inserted into the membrane of mitochondria (Li et al., 1998). In the presence of survival factors, bad is phosphorylated and sequestered in the cytosol by binding to 14-3-3 proteins. Following a death signal, Bad is dephosphorylated and then translocated to mitochondria where it interacts with, and inhibits, Bcl-XL, or Bcl-2 (Zha et al., 1996). Upon apoptotic induction, Bax is also translocated to the mitochondria although the involved molecular mechanisms remain unclear. The Bax translocation process seems to involve its dimerization and conformational change (Gross et al., 1999), which is promoted by some unidentified cytosolic factors (Nmura et al., 1999). Moreover, removal of the amino-terminal 20 amino acids of Bax enabled it to target mitochondria in vitro in the absence of an activated cytosol (Goping et al., 1998). Finally, the Bid is able to induce the oligomerization and insertion of Bax into the outer mitochondrial membrane during apoptosis (Eskes et al., 2000).
The ubiquitin/proteasome system plays an important role in the degradation of cellular proteins. This proteolytic system includes two distinct steps: ubiquitination and degradation (Antonsson et al., 1997; Chang et al., 1998). Ubiquitination is the step after which the target protein can be selectively recognized by the proteasome complex from other proteins. Ubiquitination requires a reaction cascade. First, in an energy-dependent reaction, ubiquitin is activated by, and subsequently linked to, an Ubiquitin-Activating Enzyme (E1). Second, ubiquitin is passed on from E1 to Ubiquitin-Conjugating Enzymes (E2) and often subsequently to Ubiquitin Ligases (E3). Third, ubiquitin is then conjugated to the substrate protein, catalyzed by either E2 alone or a combination of E2 with E3. Usually, multiple ubiquitin molecules are added to the substrate by the same enzyme cascade. Degradation of such multi-ubiquitinated proteins occurs on a large 26S proteasome complex in an ATP-dependent manner. The 26S proteasome complex is composed of a 20S proteasome (the catalytic core) and a pair of 700 kDa-proteasome activators (the regulatory subunit) (Antonsson et al., 1997; Chang et al., 1998).
The ubiquitin/proteasome system is involved in the regulation of apoptosis. It has been found that proteasome inhibitors, such as tripeptide aldehydes (LLnL or LLnV; Dimmeler et al., 1999) or lactacystin (a microbial metabolite; Thomas et al., 1996), induce apoptosis in human leukemia (Krajewski et al., 1994; Mackey et al., 1998) and other cell lines. It has also been found that proteasome inhibitors are able to rapidly induce apoptosis in all the human cancer cell lines tested, including leukemia, breast, prostate, lung, bone, brain and head and neck, but not in human normal fibroblasts and normal breast cells. It was also reported that proteasome inhibition is sufficient to overcome apoptotic protection by Bcl-2 or Bcr-Abl oncoprotein. Therefore, the proteasome must selectively degrade one or more cellular proteins that play an important role in apoptotic commitment. However, nature of the responsible proteasome target protein(s) remains unknown.
Regulation of apoptosis is deranged in most, if not all, human cancers (Fisher et al, 1994). Many human cancers are resistant to induction of apoptosis (Fisher et al, 1994; Harrison et al., 1995; Milner et al., 1995) at least partially due to inactivation of the tumor suppressor protein p53 (Milner et al., 1995) or overexpression of the Bcl-2 (Reed et al., 1994) or Bcr-Abl oncoprotein (Bedi et al., 1994). Indeed, higher Bcl-2/Bax ratio correlates with poor therapeutic responsiveness to radio or chemotherapy in patients with prostate (Mackey et al., 1998) or B-cell chronic lymphocytic leukemia (Pepper et al., 1998). Even reduced expression of Bax alone is associated with poor response rates to radio or chemotherapy in patients with B-cell chronic lymphocytic leukemia (Molica et al., 1998), breast (Krajewski et al., 1995), ovarian (Tai et al., 1998), cervical (Harima et al., 1998) and pediatric cancers (McPake et al., 1998). In contrast, increased levels of Bax protein, or increased ratio of Bax/Bcl-2 protein, have been found to be tightly associated with increased therapeutic response (Tai et al., 1998; Harima et al., 1998). Furthermore, it has been suggested that Bax levels also influence the prognosis of human pancreatic cancer: patients whose tumors exhibited Bax immunostaining lived significantly longer (12 months) than those whose tumors were Bax negative (5 months) (Friess et al., 1998). What determines or regulates Bax levels in human cancer cells remains unknown.
Expression of oncogenes that deregulate cell proliferation can also induce apoptosis (White, 1993; Harrington et al., 1994), indicating that oncogene expression generates a proapoptotic signal that is present in transformed cells but absent in normal cells. Indeed, most recently, it has been found that an apoptosis-promoting complex consisting caspase-9, Apaf-1 and cytochrome c regulates the process of oncogene-dependent apoptosis (Fearnhead et al., 1998). Since caspase-9, Apaf-1 and cytochrome c are also present in normal cells, it is unclear what is the missing signal in normal cells that triggers activation of the apoptosis-promoting complex.
An assay to measure some aspect of Bcl2/Bax ratio in order to select patents that are most likely to benefit from radio- or chemotherapy protocols would be useful.
A method for measuring factors that regulate Bax levels in human cancer would be useful in evalutating the prognosis of a patient with cancer. It would also be useful to develop an assay or method for determining the efficacy of prospective chemotherapeutic agents for treating cancer via altering Bax levels.
According to the present invention, there is provided an assay for determining Bax degradation activity in a patient sample. The assay includes a labeled Bax protein which is incubated with a protein extract from the sample and a detector for detecting a signal from the labeled Bax protein, whereby decreased labeled signals compared to a control indicates Bax degradation activity. Also provided by the present invention is a method for assaying a sample for Bax degradation activity for determining aggressiveness of a tumor, for screening compounds for inhibitors of Bax degradation and for determining efficacy of protease inhibitors to prevent Bax degradation including the steps of incubating the sample with a labeled Bax protein and detecting the loss of the labeled Bax protein compared to a control indicates Bax degradation activity. A method of predicting tumor aggressiveness by measuring the Bax protein level and Bax degradation level whereby low or moderate levels of Bax protein and high levels of Bax degradation activity indicate a high-grade tumor is also provided